daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear d... more daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8 % develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the ab...
Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo c... more Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centro-somes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating i...
Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesize... more Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesized nucleosomal histones H2B, H3, H4 (and possibly H2A) in chromatin are more accessible to histone acetylase in vivo than are older, pre-existing histones. Thus, when rat hepatoma cells are first pulse-labeled and then incubated in medium containing n-butyrate which blocks histone deacetylation, these newly synthesized histones become acetylated to a far greater extent than do their older homologues. As judged by its increased susceptibility to acetylation, the new chromatin matures at a surprisingly slow rate, the estimated half-time for maturation being about 35 min. Based on this data, we suggest that newly synthesized chromatin is in a relatively extended, accessible conformation, and that it slowly returns to a more compact conformation as it matures.
Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that la... more Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M....
Drosophila melanogaster has become one of the most extensively studied organisms because of its a... more Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.
Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to ... more Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.
We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm st... more We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envel...
One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h a... more One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a sm...
The scientific enterprise has greatly advanced our understanding of the natural world and has the... more The scientific enterprise has greatly advanced our understanding of the natural world and has thereby enabled the creation of countless medicines and useful devices. It has also led to behaviors that have improved lives. The public appreciates these practical benefits of science, and science and scientists are generally respected, even by those who are not familiar with how science works or what exactly it has discovered. But society may less appreciate the advantage of having everyone aquire, as part of their formal education, the ways of thinking and behaving that are central to the practice of successful science: scientific habits of mind. These habits include a skeptical attitude toward dogmatic claims and a strong desire for logic and evidence. As famed astronomer Carl Sagan put it, science is our best "bunk" detector. Individuals and societies clearly need a means to logically test the onslaught of constant clever attempts to manipulate our purchasing and political decisions. They also need to challenge what is irrational, including the intolerance that fuels so many regional and global conflicts. So how does this relate to science education? Might it be possible to encourage, across the world, scientific habits of mind, so as to create more rational societies everywhere? In principle, a vigorous expansion of science education could provide the world with such an opportunity, but only if scientists, educators, and policy-makers redefine the goals of science education, beginning with college-level teaching. Rather than only conveying what science has discovered about the natural world, as is done now in most countries, a top priority should be to empower all students with the knowledge and practice of how to think like a scientist. Scientists share a common way of reaching conclusions that is based not only on evidence and logic, but also requires honesty, creativity, and openness to new ideas. The scientific community can thus often work together across cultures, bridging political divides. Such collaborations have mostly focused on the discovery of new knowledge about the natural world. But scientists can also collaborate effectively on developing and promulgating a form of science education for all students that builds scientific habits of mind. Inquiry-based science curricula for children ages 5 to 13 have been undergoing development and refinement in the United States for more than 50 years. These curricula require that students engage in active investigations, while a teacher serves as a coach to guide them to an understanding of one of many topics. This approach takes advantage of the natural curiosity of young people, and in the hands of a prepared teacher, it can be highly effective in increasing a student's reasoning and problem-solving skills. In addition, because communication is emphasized, inquiry-based science teaching has been shown to increase reading and writing abilities. This approach to science education has been slowly spreading throughout the United States in the past decade, but it requires resources and energy on the part of school districts that are often not available. With strong support from scientists and science academies, a similar type of science education is also being increasingly implemented in France, Sweden, Chile, China, and other countries. In these efforts, catalyzed for the past 8 years by the InterAcademy Panel in Trieste, scientists are sharing resources and helping to form new bridges betweeen nations. With appropriate modifications, could such an education also help make students more rational and tolerant human beings, thereby reducing the dogmatism that threatens the world today with deadly conflict? In future editorials, I will explore the many potential advantages of inquiry-based science education. I will also discuss the barriers that must be overcome for its widespread implementation across the globe, because we may face no more urgent task if future generations are to inherit a peaceful world.
Proceedings of the National Academy of Sciences, 1972
Early events in the action of 17-β-estradiol can be studied in soluble extracts of rat uterus by ... more Early events in the action of 17-β-estradiol can be studied in soluble extracts of rat uterus by exposure of the estradiol-receptor protein to a DNA-cellulose matrix. After complexing with [ 3 H]estradiol, the 4S receptor protein binds to the DNA, and it can be eluted with buffer of high ionic strength as a more tightly binding, 5S form. This parallels the in vivo situation, where migration of the receptor to the nucleus follows addition of hormone and is concomitant with a similar increase in sedimentation rate to 5 S. In both cases, the formation of a 5S receptor requires the presence of 17-β-estradiol. The rate at which 5S receptor forms is sensitive to extract concentration in a way that suggests that this receptor is a complex created by addition of a second subunit to the hormone-binding 4S component; physical studies on both in vivo and in vitro 5S receptors also support this view. These results are interpreted in terms of a model for action of estrogen in which the hormone p...
A wake-up call How failing a PhD led to a strategy for a successful scientific career. Bruce Albe... more A wake-up call How failing a PhD led to a strategy for a successful scientific career. Bruce Alberts: 'failure' was a blessing in disguise.
Horse scents STALLIONS competing for mates judge the form of a rival male not only by listening c... more Horse scents STALLIONS competing for mates judge the form of a rival male not only by listening carefully to its whinnies but by sniffing its faeces as well (D.I. Rubenstein and M.A. Hack Evol. Ecol. 6, 254-260; 1992). Whinnies indicate dominance status, from the horse's mouth, as it were, irrespective of whether the two horses are familiar with each other or not. Faecal smell, though, reveals the identity and home turf of a particular horse, which can then be associated with the outcome of previous contests. Redundant signalling in one species is unusual, but may be a belt-and-braces solution for animals with large and overlapping ranges. The consequent saving on wasteful physical combat echoes Churchill's dictum that "to jawjaw is better than to war-war".
with the macroscopic world, these protein assemblies contain highly coordinated moving parts. Wit... more with the macroscopic world, these protein assemblies contain highly coordinated moving parts. Within each protein assembly, intermolecular collisions are not only restricted to a small set of possibilities, but reaction C depends on reaction B, which in turn depends on reac
The rolling circle DNA replication structures generated by the in vitro phage T4 replication syst... more The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or singlestranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.
W e have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polyme... more W e have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polymerase holoenzyme (polymerase plus gene 44/62 and 45 proteins) once it has been stalled by nucleotide omission. Using this assay, we determined that the dissociation of the DNA polymerase holoenzyme follows a first order decay with a half-life of 2.5 min. The long half-life resembles that expected for the holoenzyme processively synthesizing DNA on the leading strand of the replication fork. The holoenzyme dissociation rate is independent of polymerase accessory protein concentration and of ATP hydrolysis. The dissociation rate is increased if the gene 32 protein is omitted or if the primer-template region is shortened from 46 to 28 base pairs. But the rate of holoenzyme dissociation is most strikingly increased when the circular DNA template is converted to a linear form. By analogy with other well studied systems, these results support a model in which ATP hydrolysis by the 44/62 proteins serves to load a ring-like 45 protein onto the DNA. Once loaded, the 45 protein, possibly along with the 44/62 complex, acts as a sliding clamp that tethers the DNA polymerase to the template.
daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear d... more daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8 % develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the ab...
Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo c... more Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centro-somes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating i...
Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesize... more Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesized nucleosomal histones H2B, H3, H4 (and possibly H2A) in chromatin are more accessible to histone acetylase in vivo than are older, pre-existing histones. Thus, when rat hepatoma cells are first pulse-labeled and then incubated in medium containing n-butyrate which blocks histone deacetylation, these newly synthesized histones become acetylated to a far greater extent than do their older homologues. As judged by its increased susceptibility to acetylation, the new chromatin matures at a surprisingly slow rate, the estimated half-time for maturation being about 35 min. Based on this data, we suggest that newly synthesized chromatin is in a relatively extended, accessible conformation, and that it slowly returns to a more compact conformation as it matures.
Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that la... more Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M....
Drosophila melanogaster has become one of the most extensively studied organisms because of its a... more Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.
Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to ... more Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.
We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm st... more We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envel...
One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h a... more One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a sm...
The scientific enterprise has greatly advanced our understanding of the natural world and has the... more The scientific enterprise has greatly advanced our understanding of the natural world and has thereby enabled the creation of countless medicines and useful devices. It has also led to behaviors that have improved lives. The public appreciates these practical benefits of science, and science and scientists are generally respected, even by those who are not familiar with how science works or what exactly it has discovered. But society may less appreciate the advantage of having everyone aquire, as part of their formal education, the ways of thinking and behaving that are central to the practice of successful science: scientific habits of mind. These habits include a skeptical attitude toward dogmatic claims and a strong desire for logic and evidence. As famed astronomer Carl Sagan put it, science is our best "bunk" detector. Individuals and societies clearly need a means to logically test the onslaught of constant clever attempts to manipulate our purchasing and political decisions. They also need to challenge what is irrational, including the intolerance that fuels so many regional and global conflicts. So how does this relate to science education? Might it be possible to encourage, across the world, scientific habits of mind, so as to create more rational societies everywhere? In principle, a vigorous expansion of science education could provide the world with such an opportunity, but only if scientists, educators, and policy-makers redefine the goals of science education, beginning with college-level teaching. Rather than only conveying what science has discovered about the natural world, as is done now in most countries, a top priority should be to empower all students with the knowledge and practice of how to think like a scientist. Scientists share a common way of reaching conclusions that is based not only on evidence and logic, but also requires honesty, creativity, and openness to new ideas. The scientific community can thus often work together across cultures, bridging political divides. Such collaborations have mostly focused on the discovery of new knowledge about the natural world. But scientists can also collaborate effectively on developing and promulgating a form of science education for all students that builds scientific habits of mind. Inquiry-based science curricula for children ages 5 to 13 have been undergoing development and refinement in the United States for more than 50 years. These curricula require that students engage in active investigations, while a teacher serves as a coach to guide them to an understanding of one of many topics. This approach takes advantage of the natural curiosity of young people, and in the hands of a prepared teacher, it can be highly effective in increasing a student's reasoning and problem-solving skills. In addition, because communication is emphasized, inquiry-based science teaching has been shown to increase reading and writing abilities. This approach to science education has been slowly spreading throughout the United States in the past decade, but it requires resources and energy on the part of school districts that are often not available. With strong support from scientists and science academies, a similar type of science education is also being increasingly implemented in France, Sweden, Chile, China, and other countries. In these efforts, catalyzed for the past 8 years by the InterAcademy Panel in Trieste, scientists are sharing resources and helping to form new bridges betweeen nations. With appropriate modifications, could such an education also help make students more rational and tolerant human beings, thereby reducing the dogmatism that threatens the world today with deadly conflict? In future editorials, I will explore the many potential advantages of inquiry-based science education. I will also discuss the barriers that must be overcome for its widespread implementation across the globe, because we may face no more urgent task if future generations are to inherit a peaceful world.
Proceedings of the National Academy of Sciences, 1972
Early events in the action of 17-β-estradiol can be studied in soluble extracts of rat uterus by ... more Early events in the action of 17-β-estradiol can be studied in soluble extracts of rat uterus by exposure of the estradiol-receptor protein to a DNA-cellulose matrix. After complexing with [ 3 H]estradiol, the 4S receptor protein binds to the DNA, and it can be eluted with buffer of high ionic strength as a more tightly binding, 5S form. This parallels the in vivo situation, where migration of the receptor to the nucleus follows addition of hormone and is concomitant with a similar increase in sedimentation rate to 5 S. In both cases, the formation of a 5S receptor requires the presence of 17-β-estradiol. The rate at which 5S receptor forms is sensitive to extract concentration in a way that suggests that this receptor is a complex created by addition of a second subunit to the hormone-binding 4S component; physical studies on both in vivo and in vitro 5S receptors also support this view. These results are interpreted in terms of a model for action of estrogen in which the hormone p...
A wake-up call How failing a PhD led to a strategy for a successful scientific career. Bruce Albe... more A wake-up call How failing a PhD led to a strategy for a successful scientific career. Bruce Alberts: 'failure' was a blessing in disguise.
Horse scents STALLIONS competing for mates judge the form of a rival male not only by listening c... more Horse scents STALLIONS competing for mates judge the form of a rival male not only by listening carefully to its whinnies but by sniffing its faeces as well (D.I. Rubenstein and M.A. Hack Evol. Ecol. 6, 254-260; 1992). Whinnies indicate dominance status, from the horse's mouth, as it were, irrespective of whether the two horses are familiar with each other or not. Faecal smell, though, reveals the identity and home turf of a particular horse, which can then be associated with the outcome of previous contests. Redundant signalling in one species is unusual, but may be a belt-and-braces solution for animals with large and overlapping ranges. The consequent saving on wasteful physical combat echoes Churchill's dictum that "to jawjaw is better than to war-war".
with the macroscopic world, these protein assemblies contain highly coordinated moving parts. Wit... more with the macroscopic world, these protein assemblies contain highly coordinated moving parts. Within each protein assembly, intermolecular collisions are not only restricted to a small set of possibilities, but reaction C depends on reaction B, which in turn depends on reac
The rolling circle DNA replication structures generated by the in vitro phage T4 replication syst... more The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or singlestranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.
W e have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polyme... more W e have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polymerase holoenzyme (polymerase plus gene 44/62 and 45 proteins) once it has been stalled by nucleotide omission. Using this assay, we determined that the dissociation of the DNA polymerase holoenzyme follows a first order decay with a half-life of 2.5 min. The long half-life resembles that expected for the holoenzyme processively synthesizing DNA on the leading strand of the replication fork. The holoenzyme dissociation rate is independent of polymerase accessory protein concentration and of ATP hydrolysis. The dissociation rate is increased if the gene 32 protein is omitted or if the primer-template region is shortened from 46 to 28 base pairs. But the rate of holoenzyme dissociation is most strikingly increased when the circular DNA template is converted to a linear form. By analogy with other well studied systems, these results support a model in which ATP hydrolysis by the 44/62 proteins serves to load a ring-like 45 protein onto the DNA. Once loaded, the 45 protein, possibly along with the 44/62 complex, acts as a sliding clamp that tethers the DNA polymerase to the template.
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Papers by Bruce Alberts